(i) Transgenic cotton plants have been created by introducing a gene from Bacillus bacteria into American cotton plants. These transgenic cotton plants produce a toxic protein. Due to this, insect attack does not occur. As a result cotton production increased. Along with this, production costs are reduced and environmental pollution is prevented.
(ii) At present insect resistant plants like potato, apple, cotton, wheat etc. have been developed. Bt toxin gene and CpTi gene have been inserted in these plants. Insects die within 24 hours after eating the leaves of these plants.
(iii) USA company has produced caterpillar resistant cotton seeds. As a result, pest problems in cotton cultivation have been eliminated.
(iv) Cry gene isolated from Bacillus thuringiensis bacteria and introduced into plants to create insect resistant plants. Transgenic plants like Bt brinjal, Bt cotton, Bt rice, Bt corn, Bt potato, Bt apple are resistant to pests.
(v) Larvae attack of European cornbora moth causes extensive damage to bhutra and yield reduction of 40%. Transgenic bhutra has been created by introducing genes from bacteria that produce toxic proteins into bhutra plants. This bhutra is not affected by cornbora. Now there is no need to apply pesticides in Bhutra cultivation. This has increased the yield of Bhutra, reduced production costs and prevented environmental pollution.
(vi) Rhyzobium was introduced into Rhyzobium with toxin producing gene from Bacillus thuringiensis bacteria. Rhyzobium causes nodules on the roots of legumes. Bean plants with nodules can prevent attacks by harmful insects like weevils.
(vii) Bt toxin gene has been created by introducing the gene from Bacillus thuringiensis bacteria into cotton plant. Insects such as Lepidoptera, beetles, flies, mosquitoes etc. have died by eating cotton plants containing Bt toxin gene.
(viii) Sterile Insect Technique (SIT) is a modern method. It is an environmentally friendly pest control method. Males of harmful insects are sterilized by this method. As a result the new generation does not develop. This technology is being used in countries like Brazil, Japan, Philippines, Thailand, USA etc.

(i) Recombinant DNA is introduced into host Agrobacterium tumefaciens cells. The genes are then inserted into plant cells.
(ii) RNA of TMV with desired genes is introduced into tobacco plants.
Role of recombinant DNA in development of high yielding varieties
High yielding varieties have been created by introducing desired genes from wild species into crop plants. Genes producing beta carotene and iron from daffodil plant have been introduced into rice, wheat, bhutra, soybean, potato, tomato, papaya, rye, sunflower, pear, grape etc. to create high yielding varieties.

(i) Calcium chloride: Cells are first incubated in cold CaCl2 solution and heat-shocked to introduce recombinant DNA into host cells.
(ii) Liposome: Recombinant DNA is first incorporated into artificial vesicles. The vesicle is then attached to the cell membrane. The recombinant DNA is then introduced into the host cell.

Recombinant DNA is first incubated with artificial vesicles. The vesicle is then attached to the cell membrane. The recombinant DNA is then introduced into the host cell.

(i) Electroporation: Pores are created in the host cell membrane by an electric field. Recombinant DNA is introduced into the host cell through this pore.
(ii) Micro-injection: The host cell is held by a micropipette and the recombinant DNA is injected into the host cell by a fine needle.
(iii) Biolistics: Recombinant DNA is placed on the surface of a metal particle (gold) and that particle is gunshotted into the plant cell.

Recombinant DNA is placed on top of a metal particle (gold) and the particle is gunshotted into the plant cell.

The host cell is captured with a micropipette and the recombinant DNA is injected into the host cell with an ultrafine needle.

A hole is created in the host cell membrane by the electric field. Recombinant DNA is introduced into the host cell through this pore.

1. Target DNA selection and separation (Target DNA selection): First the target DNA is selected. While selecting the desired DNA, one should keep in mind that it should be healthy, strong, disease free and of superior breed. Selected cells are first lysed slightly. Cell membranes are broken down using enzymes like lysozyme (bacterial cells), chitinase (fungal cells), cellulase (plant cells). The lysed cells are then centrifuged into test tubes. A quantity of cesium chloride solution is introduced into a centrifuge test tube. The solution is then centrifuged. As a result, a band of DNA is formed on the test tube. DNA is mixed with other components to form a homogenate. DNA is separated from the homogenate using enzymes such as protease (protein), ribonuclease (RNA), amylase (sugar), lipase (fat). The purified DNA is then precipitated as a thread by immersion in a cold ethanol solution. The desired DNA is selected from the degraded DNA.
2. Host selection: The host is selected to carry the required part of the desired DNA. In this case, the bacterium Agrobacterium tumefaciens works as a good carrier. The required part of the desired DNA is attached to the plasmid DNA located in the cytoplasm of this bacterium. In some cases carriers such as cosmids, phagemids, artificial chromosomes etc. are used.
3. Excision of the desired DNA at a specific location: Several sections are cut from the desired DNA by applying restriction enzymes. Corresponding segments are also excised from the carrier plasmid DNA using the same enzyme. This process is called restriction digestion. Restriction enzymes cleave double-stranded DNA to form single-stranded ends. It is called sticky end.
4. Placement of the desired DNA molecule into the carrier plasmid DNA molecule: The desired fragment is separated from the DNA fragments by gel electrophoresis. The desired DNA fragment is then picked up on a nylon screen by Southern blotting. The required gene is then identified using a radioactive probe. The identified DNA is ligated to plasmid DNA with ligase enzymes. Recombinant DNA is produced by ligation of the desired DNA with the carrier plasmid DNA.
5. Introduction of Recombinant DNA into the Host: The host is selected to carry the recombinant DNA. In this case E. coli bacteria act as host. Recombinant DNA is introduced into host cells. But under normal conditions bacteria do not accept other plasmids. If the culture medium in which the bacterium is grown is heated and a suitable environment is created by adding calcium, the bacterium takes up another plasmid. The process of introducing recombinant DNA into bacterial cells is called transformation. Besides, recombinant DNA is introduced into host cells through conjugation, microinjection, liposome, electroporation etc.
6. Evaluation of expression of recombinant DNA: It is checked whether the work of making recombinant DNA has been done correctly. This test is done by genetic probe method. Selectable marker of plasmid DNA is used in this process. Antibiotic resistance genes are inserted into the recombinant DNA. The host bacteria are then grown in culture medium. Bacteria into which the antibiotic resistance gene was inserted grew in the culture medium and formed colonies. From this, it can be understood that the work of making recombinant UghA has been done correctly.
7. Introduction of Recombinant DNA into Plant Body: The prepared recombinant DNA is introduced into the desired plant body in tissue culture process. Later new plants are obtained from those cells. Such plants are called transgenic plants.

Check if the recombinant DNA has been made correctly. This test is done by genetic probe method. Selectable marker of plasmid DNA is used in this process. Antibiotic resistance genes are inserted into the recombinant DNA. The host bacteria are then grown in culture medium. Bacteria into which the antibiotic resistance gene was inserted grew in the culture medium and formed colonies. From this, it can be understood that the work of making recombinant UghA has been done correctly.